PLCE1基因多态性影响基因表达与蛋白修饰及参与胃癌形成的分子机制研究

Statistic shown that both the morbidity and mortality of gastric cancer are ranked number one in Chinese cancer patients. According to a hospital based case control study in Sequenom MassARRAY platform carried out in our Lab, three single nucleotide polymorphisms (SNPs) in phospholipase C epsilon 1 (PLCE1) gene were identified significantly associated with gastric cancer in northwest Chinese populations. Amonng them, two nonsynonymous mutations of rs3765524C>T and rs2274223A>G have caused amino acids mutation of Thr1777Ile and His1927Arg respectively, strongly suggest that PLCE1 mutation may play important role in gastric cancer development. PLCE1 is a new member of phospholipase C family. Bioinformatics predicts that mutations in rs3765524 and rs2274223 might have some enfluences on its protein modification, such as phosphorylation or methylation. PLCE1 may interact to the catalytic subunit of DNA-dependent serine/threonine protein kinase (DNA-PK). According to references, we also found that PLCE1 could potentially regulate RAS-BRAF signal pathway by protein interaction, which has been certificated as the key checkpoints in gastric cancer. In this study, we will explore the relationship between the PLCE1 SNPs and the development of gastric cancer. To elucidate why and how the SNPs regulate PLCE1 gene expression and/or protein modification. To identify proteins interacte with PLCE1, to characterize its signal passway with methods of gene transfection, immunoprecipitation, mass spectrography analysis and so on. Finally to understand the mechanism of cell prolification and differentiation in gastric cancer development. And to provide potential candidate targets and new strategies for the clinical diagnosis and therapy in gastric cancer.

胃癌是中国癌症发病和死亡的首位肿瘤。我课题组应用MassARRAY平台进行的600例病例-对照研究发现PLCE1基因多态性与我国西北地区汉族人群的胃癌发病密切相关，其中rs3765524和rs2274223 的SNP发生在可修饰的氨基酸上，提示该突变可能影响PLCE1的结构与功能。PLCE1是磷酯酶C同工酶，结构与功能尚未明确。生物信息学预测PLCE1可能具有多种蛋白修饰，具有结合DNA依赖的蛋白激酶催化亚基结构域。文献提示PLCE1可能参与RAS-BRAF信号通路的调控，在肿瘤发生中发挥重要功能。本课题拟通过分析PLCE1基因多态性与胃癌分化分期的关系，采用体外基因转染、免疫共沉淀、质谱分析等方法研究PLCE1基因多态性如何影响基因表达和蛋白修饰，筛选相互作用蛋白，发现相关信号通路，为PLCE1参与胃癌形成的分子机制提出新的实验依据，为胃癌的临床诊断和治疗提供新的候选靶位和策略。

研究背景：胃癌是严重威胁我国人民健康和生命的疾病之一，具有高发病率、高死亡率，病因复杂，缺乏预警手段等特点。胃癌早期诊断、治疗特异性生物标志物研究具有重要意义。单核苷酸多态性（SNP）作为遗传标志物被广泛应用于胃癌发病风险及预后评价的关联分析中。磷脂酶Cε1（PLCE1）SNP与胃癌风险的相关性及其分子机制亟待阐明。.研究结果：本研究以Massarray 质谱检测平台为主要技术手段，筛选发现了PLCE1基因三个SNP位点（rs10509670、 rs3765524、rs2274223）与我国西北汉族人群胃癌易感性显著相关；LD分析发现2个与胃癌风险相关联的单体型；表达分析显示PLCE1差异表达于胃癌及癌旁组织；rs3765524和rs2274223基因型影响PLCE1基因表达，minor型PLCE1表达明显高于野生型；体外克隆测序获得了新的PLCE1 异构体；minor型PLCE1 过表达可促进细胞增殖、迁移和克隆形成，促进胃癌细胞的EMT过程，与胃癌细胞对化疗药物(HCPT, DOXO, 5-FU)的耐药性相关；耐药性的形成与PLCE1促进DNA-PK表达、促进高尔基体重塑与分散相关，联合使用DNA-PK抑制剂可以逆转细胞耐药。.研究结论：本研究在我国西北汉族人群中进行胃癌相关生物标志物研究，发现了PLCE1 基因SNP与胃癌的易感性相关，阐明了SNP影响PLCE1基因表达以及与DNA-PK相互作用在化疗药物耐药中的作用，为逆转化疗耐药提供了新的策略。